The present invention relates principally to novel compounds which are related to the group B streptogramins, and to a process for preparing streptogramins by mutasynthesis. It also relates to novel genes which are involved in the biosynthesis of precursors of the group B streptogramins, and to their uses.
The streptogramins form a homogeneous group of antibiotics consisting of an association of two types of chemically different molecules; on the one hand polyunsaturated macrolactones (group A components) and, on the other hand, depsipeptides (group B components). This group comprises numerous antibiotics which are known under different names according to their origin and includes pristinamycins, mikamycins and virginiamycins (Cocito 1979, 1983).
The A and B components have a synergistic antibacterial activity which can amount to 100 times that of the separate components and which, contrary to that of each component, is bactericidal (Cocito 1979). This activity is more particularly effective against Grai-positive bacteria such as Staphylococci and Streptococci (Cocito 1979, Videau 1982). Components A and B inhibit protein synthesis by binding to the 50S subunit of the ribosome (Cocito 1979; Di Giambattista et al., 1989).
While knowledge of the routes by which each of the components is biosynthesized still remains partial to date, earlier studies, presented in Patent Application PCT/FR93/0923, have made it possible to identify several proteins, and the corresponding structural genes, which are involved in the biosynthesis of the two types of component.
Two parts can be distinguished in the process for biosynthesizing group B streptogramins:
1) Biosynthesis of the precursors, or their analogues, of the macrocycle: 3-hydropicolinic acid, L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine, L-pipecolic acid and L-phenylglycine.
2) Formation of the macrocycle from the precursors listed above, from L-threonine and from L-proline, or their analogues, with (a) possible subsequent modification(s) of the peptide N-methylation, epimerisation, hydroxylation and oxidation type.
Patent Application PCT/FR93/0923 relates, in particular, to the enzymes which catalyze incorporation of the precursors into the peptide chain of B streptogramins in the process of elongation, and also to their structural genes. These results have demonstrated the non-ribosomal peptide synthesis character of the type B components.
The present invention relates, more particularly, to novel compounds which are related to group B streptogramins and, more precisely, to novel compounds of the pristinamycin I family (FIGS. 1 and 2), termed PI below, or of the virginiamycin S family (FIG. 3).
The major constituent of the I pristinamycins (PI) is PIA (FIG. 1), which represents approximately 94% of the PI, with the remaining approximately 6% being represented by minor constituents of the depsipeptide (PIB, to PII) whose structures are depicted in FIG. 2. PI results essentially from the condensation of amino acids, certain of which are essential for protein synthesis (threonine and proline) and others of which are novel and themselves considered to be secondary metabolites (L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine (DMPAPA), L-pipecolic acid and L-phenylglycine for PIA), and also of an aromatic precursor, 3-hydroxypicolinic acid.
The virginiamycin S derivatives result from condensation of the same acids as in the case of PI, apart from 4-DMPAPA, which is replaced by a phenylalanine (see FIG. 3).
Production of these different compounds by biosynthesis therefore requires preliminary synthesis, by a producer strain, of the novel precursors identified above.
The present invention results specifically from a novel process for preparing streptogramins which employs, as a strain for producing streptogramins, a microorganism strain which is mutated so as to alter the biosynthesis of the precursors of the group B streptogramins. According to this process, the said mutant strain is cultured in a medium which is supplemented with a novel precursor which is different from the precursor whose biosynthesis is altered. Unexpectedly, this results in the production of novel compounds which are related to the group B streptogramins and which are of value in the therapeutic sphere.
More precisely, the present invention relates to novel compounds which are represented by the general formula I: 
in which:
R2 and R4 represent, independently of each other, a hydrogen atom or a methyl group,
R3 represents a hydrogen atom or a hydroxyl group,
X represents a CO, CHOH or CH2 group, and
R1 represents: 
with
for the meta derivatives:
A, C, D and E representing a hydrogen atom, and
B being able to represent
a halogen, and preferably a fluorine atom,
a monoalkylamino or dialkylamino group, with alkyl preferably representing a methyl or ethyl group,
an ether group; more particularly an OR group with R being preferably selected from among the methyl, ethyl, trifluoromethyl and allyl groups,
a thioether group, preferably represented by an alkylthio group with alkyl preferably representing a methyl group,
a C1 to C3 alkyl group, or
a trihalogenomethyl group, preferably trifluoromethyl
for the para derivatives
A, B, D and E representing a hydrogen atom, and C being able to represent:
a halogen,
an NR1R2 group with R1 and R2 representing, independently of each other, a group selected from among
hydrogen,
a straight-chain or branched C1 to C4 alkyl group where, when one of the substituents R1 or R2 represents a methyl group, the other necessarily represents an ethyl group,
an alkyl-cycloalkylmethyl group with a C3 to C4 cycloalkyl,
an optionally substituted C3 to C4 cycloalkyl group,
a straight-chain or branched C1 to C4 alkenyl group where, when one of the substituents R1 or R2 represents an alkenyl group, the other is different from a methyl group or a C3 to C6 cycloalkyl,
a substituted or unsubstituted N-pyrrolidinyl group,
an ether group; preferably an OR group with R preferably being selected from among the methyl and ethyl groups, where appropriate substituted by a chlorine atom, or trifluoromethyl and alkenyl groups
a thioether group, preferably represented by an alkylthio group with alkyl preferably representing a C1 to C3 alkyl group,
an acyl or alkoxycarbonyl group and, more particularly, a COR group with R preferably representing a C1 to C3 alkyl group or a C1 to C3 alkoxy group,
a C1 to C6 alkyl group which is straight-chain or branched and which is preferably selected from among the methyl, isopropyl and tert-butyl groups,
an alkylthiomethyl group and, more preferably, a CH2SR group with R preferably representing a C1 to C3 alkyl group,
an aryl group, preferably a phenyl group, or
a trihalogenomethyl group, preferably trifluoromethyl
for the meta-para disubstituted derivatives:
A, D and E representing a hydrogen atom, and
B being able to represent:
a halogen, preferably a fluorine atom,
a monoalkylamino or dialkylamino group with alkyl preferably representing a methyl or ethyl group,
an ether group and preferably an OR group with R preferably selected from among the methyl, ethyl and trifluoromethyl groups,
a thioether group and preferably alkylthio with alkyl preferably representing an ethyl group, or
a C1 to C3 alkyl group, and
C being able to represent:
a halogen, preferably a fluorine atom,
an amino, monoalkylamino or dialkylamino group with alkyl preferably representing a methyl group with the proviso that B is different from a bromine or chlorine atom, or a substituted or unsubstituted allyl group,
an ether group and preferably an OR group with R preferably selected from among the methyl, ethyl and trifluoromethyl groups,
a thioether group and preferably an alkylthio group with alkyl preferably representing a methyl group,
a C1 to C6 alkyl group, or
a trihalogenomethyl group, preferably trifluoromethyl, and
for the ortho-para disubstituted derivatives:
B, E and D representing a hydrogen atom and A and C a methyl group.
The following may be more particularly mentioned as preferred compounds:
4xcex6-methylthio-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-methylthio-de(4xcex6-dimethylamino)pristinamycin IH,
5xcex3-hydroxy-4xcex6-methylthio-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex6-methyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-methyl-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex6-methoxy-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-methoxycarbonyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-chloro-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-bromo-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-bromo-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex6-iodo-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-iodo-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex6-trifluoromethyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-trifluoromethyl-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex6-tert-butyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-isopropyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-isopropyl-de(4xcex6-dimethylamino)pristinamycin IE,
4xcex5-methylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex5-methoxy-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex5-methoxy-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex5-fluoro-4xcex6-methyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-amino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-diethylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-allylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-diallylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-allylethylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethylpropylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethylisopropylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethylmethylcyclopropylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-(1-pyrrolidinyl)-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-trifluoromethoxy-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-allyloxy-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethoxy-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethylthio-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-methylthiomethyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-(2-chloroethoxy)-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-acetyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethyl-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex6-ethyl-de(4xcex6-dimethylamino)pristinamycin IH,
4xcex5-dimethylamino-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex5-methylthio-de(4xcex6-dimethylamino)pristinamycin IA,
4xcex5-ethoxy-de(4xcex6-dimethylamino)pristinamycin IA.
The present invention is also directed towards a process which is particularly useful for preparing the compounds of the general formula I.
More precisely, it relates to a process for preparing streptogramins, characterized in that it employs a streptogramin-producing microorganism strain which possesses at least one genetic modification which affects the biosynthesis of a precursor of the group B streptogramins, and in that the said mutant strain is cultured in a culture medium which is appropriate and which is supplemented with at least one novel precursor which is different from that whose biosynthesis is altered, and in that the said streptogramins are recovered.
The strains which are employed within the scope of the present invention are therefore strains which produce streptogramins and which are mutated. The genetic modification(s) can be located either within one of the genes which is involved in the biosynthesis of the said precursors or outside the coding region, for example in the regions responsible for the expression and/or the transcriptional or post-transcriptional regulation of the said genes, or in a region belonging to the transcript containing the said genes.
According to one particular embodiment of the invention, the mutant strains possess one or more genetic modifications within at least one of their genes which is/are involved in the biosynthesis of the group B streptogramin precursors.
This or these genetic modification(s) alter(s) the expression of the said gene, that is render(s) this gene, and, as the case may be, another of the genes involved in the biosynthesis of the precursors, partially or totally incapable of encoding the natural enzyme which is involved in the biosynthesis of at least one precursor. The inability of the said genes to encode the natural proteins may be manifested either by the production of a protein which is inactive due to structural or conformational modifications, or by the absence of production, or by the production of a protein having an altered enzymatic activity, or else by the production of the natural protein at an attenuated level or in accordance with a desired mode of regulation. The totality of these possible manifestations is expressed by an alteration of, or perhaps a blockage in, the synthesis of at least one of the group B streptogramin precursors.
The genes which are capable of being mutated within the scope of the present invention are preferably the genes which are involved in the biosynthesis of the following precursors: L-2-aminobutyric acid, 4-dimethylamino-L-phenylalanine (DMPAPA), L-pipecolic acid, L-phenylglycine and/or 3-hydroxypicolinic acid (3-HPA).
These genes are more preferably the papA (SEQ ID NO: 14), papM (SEQ ID NO: 16), papB (SEQ ID NO: 4), papC (SEQ ID No. 2), hpaA (SEQ ID NO: 12), snbF (SEQ ID NO: 9), and pipA (SEQ ID NO: 7) genes described below.
The papA and papM genes have already been described in Patent Application PCT/FR93/0923. They are present on the cosmid pIBV2. The papA pap gene appears to correspond to a gene for biosynthesizing 4-amino-L-phenylalanine from chorismate. The 4-amino-L-phenylalanine is then dimethylated by the product of the papM gene, an N-methyltransferase, in order to form 4-dimethylamino-L-phenylalanine, DMPAPA, which is then incorporated into pristinamycin IA. These two genes are more particularly involved, therefore, in the synthesis of the precursor termed DMPAPA.
The other genes, papB, papC, pipA, snbF and hpaA, have been identified and characterized within the scope of the present invention. They are grouped together with the snbA, papA and papM genes on a chromosomal region of approximately 10 kb (FIG. 7).
The sequence homologies demonstrated for the PapB (SEQ ID NO: 5) and PapC (SEQ ID NO: 3) proteins show that these proteins are also involved, jointly with the PapA (SEQ ID NO: 15) and PapM (SEQ ID NO: 17) proteins, in the biosynthesis of the DMPAPA precursor. The two corresponding novel genes, papB and papC, were isolated and identified by subcloning which was carried out using cosmid pIBV2, described in Patent Application PCT/FR93/0923, and a plasmid, pVRC900, which is derived from pIBV2 by means of a HindIII deletion and is also described in Patent Application PCT/FR93/0923.
The comparison of the protein encoded by the papC gene with the protein sequences contained in the Genpro library shows a 27% homology with the region which is involved in the prephenate dehydrogenase activity of the bifunctional TyrA proteins of E. coli (Hudson and Davidson, 1984) and Erwinia herbicola (EMBL data library, 1991). This region of TyrA catalyses aromatization of the prephenate to form 4-hydroxyphenylpyruvate in the biosynthesis of tyrosine. A similar aromatization, which proceeds from 4-deoxy-4-aminoprephenate and leads to 4-aminophenyl-pyruvate is very probably involved in the synthesis of DMPAPA. It would be catalysed by the PapC protein (SEQ ID No. 2).
PapB possesses a 24 to 30% homology with the region which is involved in the chorismate mutase activity of the TyrA and PheA bifunctional proteins of E. coli, (Hudson and Davidson, 1984) and of the TyrA protein of Erwinia herbicola. This region catalyses isomerization of the chorismate to form prephenate in the biosynthesis of tyrosine and of phenylalanine. The PapB protein (SEQ ID No. 3) is probably involved in a similar isomerization which proceeds from 4-deoxy-4-aminochorismate and leads to 4-deoxy-4-aminoprephenate in the synthesis of DMPAPA.
The pipA, snbF and hpaA genes have been located in the regions which are contained between the snbA gene, which encodes 3-hydroxypicolinic acid AMP ligase and is described in Patent Application PCT/FR93/0923, and the papA or snbR genes. They were located accurately by means of subcloning, which was carried out using the plasmid pVRC900 and the cosmid pIBV2, which are described in Patent Application PCT/FR93/0923.
On comparing the protein encoded by the hpaA gene and the protein sequences contained in the Genpro library, a homology of from 30 to 40% was detected with a group of proteins which are probably involved (Thorson et al., 1993) in the transamination of intermediates in the biosynthesis of various antibiotics (DnrJ, EryCl, TylB, StrS and PrgL). Synthesis of the 3-HPA precursor, which appears to derive from lysine by another route than that of cyclodeamination (see examples 1-2 and 2-1), probably requires a transamination step which can be catalyzed by the product of this gene termed hpaA (SEQ ID NO: 12). Furthermore, the results of mutating this gene demonstrate unequivocally that it is involved in the synthesis of the 3-HPA precursor.
Comparison of the product encoded by the gene termed pipA with the protein sequences contained in the Genpro library shows a 30% homology with the ornithine cyclodeaminase of Agrobacterium tumefaciens (Schindler et al., 1989). This enzyme is involved in the final step of the catabolism of octopine; it converts L-ornithine into L-proline by means of cyclodeamination. Authors have demonstrated, by means of incorporating labelled lysine, that 4-oxopipecolic acid and 3-hydroxypicolinic acid, which are found both in PIA and in virginiamycin S1, derived from lysine (Molinero et al., 1989, Reed et al., 1989). Cyclodeamination of lysine, in a similar manner to that described for ornithine, would lead to the formation of pipecolic acid. Taking this hypothesis into account, this product was termed PipA (SEQ ID NO: 7). The results of mutating the pipA gene, presented in the examples below, demonstrate that it is involved solely in the synthesis of pipecolic acid. It is noted, in particular, that this mutation has no effect on the biosynthesis of 3-hydroxypicolinic acid, which is also derived from lysine and of which pipecolic acid could have been a precursor.
Finally, on comparing the product of the gene termed snbF with the protein sequences contained in the Genpro library, a 30 to 40% homology was noted with several hydroxylases of the cytochrome P450 type, which are involved in the biosynthesis of secondary metabolites (Omer et al., 1990; Trower et al., 1992). Several hydroxylations can be envisaged in the biosynthesis of the precursors of pristinamycin I, in particular in the biosynthesis of 3-HPA (hydroxylation of picolinic acid at the 3 position) and of 4-oxopipecolic acid (hydroxylation of pipecolic acid at the 4 position). The corresponding protein was termed SnbF (SEQ ID NO: 9).
The results of mutating the pipA gene, with polar effects on the expression of the snbF gene, demonstrate the involvement of the snbF gene in the hydroxylation of the pipecolic acid residue of group B streptogramins. The expression of the snbF gene is thus altered by the expedient of effecting a genetic modification of the pipA gene.
Preferentially, the genetic modification(s) render(s) the said gene partially or totally incapable of encoding the natural protein.
Genetic modification should be understood to mean, more particularly, any suppression, substitution, deletion, or addition of one or more bases in the gene(s) under consideration. Such modifications may be obtained in vitro (on the isolated DNA) or in situ, for example, by means of genetic engineering techniques, or else by exposing the said microorganisms to a treatment using mutagenic agents. Examples of mutagenic agents which may be cited are physical agents such as high energy rays (X, xcex3, ultraviolet, etc. rays), or chemical agents which are able to react with different functional groups of the DNA bases, and, for example, akylating agents [ethyl methanesulphonate (EMS), N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine, and N-nitroquinoline-1-oxide (NQO)], bialkylating agents, intercalating agents, etc. Deletion is understood to mean any suppression of a part or all of the gene under consideration. This deletion can, in particular, be of a part of the region encoding the said proteins, and/or of all or part of the promoter region for transcription or translation, or else of the transcript.
The genetic modifications may also be obtained by means of gene disruption, for example using the protocol initially described by Rothstein [Meth. Enzymol. 101 (1983) 202] or, advantageously, by means of double homologous recombination. In this case, the integrity of the coding sequence will preferentially be disrupted in order to permit, if need be, replacement, by means of homologous recombination, of the wild-type genomic sequence with a non-functional or mutant sequence.
According to another option of the invention, the genetic modifications can consist of placing the gene(s) encoding the said proteins under the control of a regulated promoter.
The mutant microorganism strains according to the present invention may be obtained from any microorganism which produces streptogramins (cf. Table V). According to one particular embodiment of the invention, the mutant strain is a strain which is derived from S. pristinaespiralis and, more particularly, from S. pristinaespiralis SP92.
Mutant strains which are preferred within the scope of the present invention and which may more particularly be mentioned are the strain SP92::pVRC508, which is mutated in the biosynthesis of the DMPAPA precursor by disrupting the papA gene by means of simple crossing over, or else, more preferably, the strain SP212, which is mutated in the biosynthesis of the DMPAPA cursor by disrupting the papA gene by means of double homologous recombination. These strains no longer produce PI unless they are supplemented with the DMPAPA precursor. Unexpectedly, when a novel precursor, which is different from DMPAPA and which is capable, after, in this case, metabolization, of being incorporated by PI synthetase III (SnbD protein which is responsible for incorporating L-proline and DMPAPA residues) is added to the production medium, these two strains then become able to produce novel I pristinamycins or virginiamycins, or else mainly to produce a component which is normally a minor component of PI, in particular PIB (FIG. 2).
Two other mutant strains have been prepared within the scope of the present invention. These are, respectively, the strain SP92pipA::xcexa9amR, in which the pipA gene is disrupted by homologous recombination, and the strain SP92hpaA::xcexa9amR, in which the hpaA gene is disrupted. While strain SP92pipA::xcexa9amR no longer produces PI under standard fermentation conditions it strongly produces, in the presence of L-pipecolic acid, a component, which was initially a minor component among the B streptogramin components, in which 4-oxopipecolic acid is replaced by L-pipecolic acid. While strain S. pristinaespiralis SP92hpaA::xcexa9amR no longer produces PI under standard fermentation conditions, it is able to produce novel group B streptogramins in the presence of novel precursors.
By supplementing the medium for culturing mutant strains according to the invention with at least one novel precursor, it turns out that it is possible to orient biosynthesis either towards novel streptogramins, or towards a minor form of the streptogramins, or else to favour formation of one of the streptogramins.
The precursors which are employed within the scope of the present invention can be derivatives or analogues of amino acids and, more particularly of phenylalanine, as well as organic acids and, in particular, alpha-cetocarboxylic acids and, more particularly, derivatives of phenylpyruvic acid.
Naturally, the novel precursor is such that it complements the alteration or blockage, which is induced in accordance with the invention, within the biosynthesis of one of the natural precursors of the group B streptogramins and leads to the synthesis of streptogramins. According to one particular embodiment of the invention, this novel precursor is selected such that it is related to the precursor whose biosynthesis is altered. Thus, in the specific case of the mutant which is blocked in the biosynthesis of DMPAPA, the novel precursor is preferably a derivative of phenylalanine.
The following may, in particular, be cited as precursors which are suitable for the invention:
Phenylalanine, 4-dimethylaminophenylalanine, 4-methylaminophenylalanine, 4-aminophenylalanine, 4-diethylaminophenylalanine, 4-ethylaminophenylalanine, 4-methylthiophenylalanine, 4-methylphenylalanine, 4-methoxyphenylalanine, 4-trifluoromethoxyphenylalanine, 4-methoxycarbonylphenylalanine, 4-chlorophenylalanine, 4-bromophenylalanine, 4-iodophenylalanine, 4-trifluoromethylphenylalanine, 4-tert-butylphenylalanine, 4-isopropylphenylalanine, 3-methylaminophenylalanine, 3-methoxyphenylalanine, 3-methylthiophenylalanine, 3-fluoro-4-methylphenylalanine, L-pipecolic acid, 4-tert-butylphenylpyruvic acid, 4-methylaminophenylpyruvic acid, 2-naphthylphenylalanine, 4-fluorophenylalanine, 3-trifluorophenylalanine, 3-ethoxyphenylalanine, 2,4-dimethylphenylalanine, 3,4-dimethylphenylalanine, 3-methylphenylalanine, 4-phenylphenylalanine, 4-butylphenylalanine, 2-thienyl-3-alanine, 3-trifluoromethylphenylalanine, 3-hydroxyphenylalanine, 3-ethylaminophenylalanine, 4-allylaminophenylalanine, 4-diallylaminophenylalanine, 4-allylethylaminophenylalanine, 4-ethylpropylaminophenylalanine, 4-ethylisopropylaminophenylalanine, 4-ethylmethylcyclopropylaminophenylalanine, 4-(1-pyrrolidinyl)phenylalanine, 4-O-allyltyrosine, 4-O-ethyltyrosine, 4-ethylthiophenylalanine, 4-ethylthiomethylphenylalanine, 4-O-(2-chloroethyl)tyrosine, 4-acetylphenylalanine, 4-ethylphenylalanine, 3-dimethylaminophenylalanine, 3-ethoxyphenylalanine, 3-fluoro-4-methylphenylalanine and 4-aminomethylphenylalanine.
Among these precursors, 4-trifluoromethoxyphenylalanine, 3-methylaminophenylalanine, 3-methylthiophenylalanine, 3-fluoro-4-methylphenylalanine, 4-methylaminophenylpyruvic acid, 3-ethoxyphenylalanine, 4-allylaminophenylalanine, 4-diallylaminophenylalanine, 4-allylethylaminophenylalanine, 4-ethylpropylaminophenylalanine, 4-ethylisopropylaminophenylalanine, 4-ethylmethylcyclopropylaminophenylalanine, 4-(1-pyrrolidinyl)phenylalanine, 4-ethylthiomethylphenylalanine, 4-O-(2-chloroethyl)tyrosine, 3-dimethylaminophenylalanine and 3-ethylaminophenylalanine are novel and were prepared and characterized within the scope of the present invention. They are found to be particularly useful for preparing streptogramins according to the invention.
The claimed process turns out to be particularly adventageous for preparing novel group B streptogramins or else for favoring formation of particular streptogramins. As such, it is particularly useful for preparing PIB.
The present invention also relates to a nucleotide sequence which is selected from among:
(a) all or part of the genes papC (SEQ ID No. 2), papB (SEQ ID NO: 4), pipA (SEQ ID NO: 7), snbF (SEQ ID NO: 9) and hpaA (SEQ ID NO: 12),
(b) sequences which hybridize with all or part of the (a) genes, and
(c) sequences which are derived from (a) and (b) sequences on account of the degeneracy of the genetic code.
In the particular case of the hybridizing sequences according to (b), these sequences preferably encode a polypeptide which is involved in the biosynthesis of the streptogramins.
Still more preferably, the invention relates to the nucleotide sequences which are represented by the genes papC (SEQ ID No. 2), papB (SEQ ID NO: 4), pipA (SEQ ID NO: 7), snbF (SEQ ID NO: 9), and hpaA (SEQ ID NO: 12).
The invention furthermore relates to any recombinant DNA which encompasses a papC (SEQ ID No. 2), papB (SEQ ID NO: 4), pipA (SEQ ID NO: 7), snbF (SEQ ID NO: 9) or hpaA (SEQ ID NO: 12) gene.
Naturally, the nucleotide sequences defined above can be part of a vector of the expression vector type, which can be an autonomously replicating vector, an integrated vector or a suicide vector. The present invention is also directed to these vectors as well as to any use of a sequence according to the invention or of a corresponding vector for, in particular, preparing metabolites of interest. It furthermore relates to any polypeptide which results from the expression of a claimed sequence.
The present invention also relates to any mutated S. pristinaespiralis strain which possesses at least one genetic modification within one of the papC (SEQ ID No. 2), papB (SEQ ID NO: 4), pipA (SEQ ID NO: 7), snbF (SEQ ID NO: 9) and hpaA (SEQ ID NO: 12) genes, and, more preferably, to strains SP92pipA::xcexa9amR and SP92hpaA::xcexa9amR, as well as any S. pristinaespiralis strain, such as SP212, which possesses a genetic modification which consists of a disruption of the papA gene by means of double homologous recombination.
Combinations of a component of the group A streptogramins and of a compound of the general formula I, according to the invention, constitute compositions which are particularly advantageous in the therapeutic sphere. They are employed, in particular, for treating ailments which are due to Gram-positive bacteria (of the genera Staphylococci, Streptococci, Pneumococci and Enterococci) and Gram-negative bacteria (of the genera Haemophilus, Gonococci, Meningococci). Thus, the compounds according to the invention have a synergistic effect on the antibacterial action of pristinamycin IIB on Staphylococcus aureus IP8203 in mice in vivo, at oral doses which are principally between 30 mg/kg and 100 mg/kg, when they are combined in PI/PII proportions of the order of 30/70.
The present invention extends to any pharmaceutical composition which contains at least one compound of the general formula I which is or is not combined with a group A streptogramin.
The examples appearing below are presented by way of illustrating the present invention and do not limit it.